The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3(-) transport protein OsNAR2.1 in NO3(-)-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3(-) concentrations, but not under low NH4(+) concentrations. (15)N-labelling NO3(-) supplies (provided at concentrations of 0-10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3(-) concentrations resulted from impaired NO3(-) uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3(-) supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3(-)-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3(-) uptake and NO3(-)-signalling.